Blaney & Howard's Basic & Applied Concepts of Blood Banking and Transfusion Practices (3rd Edition) PDF

BASIC & APPLIED CONCEPTS of BLOOD BANKING and TRANSFUSION PRACTICES Third Edition YOU’VE JUST PURCHASED MORE THAN A TEXTBOOK ACTIVATE THE COMPLETE LEARNING EXPERIENCE THAT COMES WITH YOUR BOOK BY REGISTERING AT http://evolve.elsevier.com/Blaney/bloodbank/ Evolve Resources...

BASIC & APPLIED CONCEPTS of BLOOD BANKING and TRANSFUSION PRACTICES Third Edition YOU’VE JUST PURCHASED MORE THAN A TEXTBOOK ACTIVATE THE COMPLETE LEARNING EXPERIENCE THAT COMES WITH YOUR BOOK BY REGISTERING AT http://evolve.elsevier.com/Blaney/bloodbank/ Evolve Resources for Basic & Applied Concepts of Blood Banking and Transfusion Practices offers the following features: Case Studies Laboratory Manual REGISTER TODAY! BASIC & APPLIED CONCEPTS of BLOOD BANKING and TRANSFUSION PRACTICES Third Edition Kathy D. Blaney, MS, BB(ASCP)SBB Tissue Typing Laboratory Florida Hospital Orlando, Florida; LifeSouth Community Blood Centers Gainesville, Florida Paula R. Howard, MS, MPH, MT(ASCP)SBB Community Blood Centers of Florida A Division of OneBlood, Inc. Lauderhill, Florida 3251 Riverport Lane St. Louis, Missouri 63043 BASIC & APPLIED CONCEPTS OF BLOOD BANKING AND TRANSFUSION PRACTICES ISBN: 978-0-323-08663-9 Copyright © 2013 by Mosby, an imprint of Elsevier Inc. Copyright © 2009, 2000 by Mosby, Inc., an affiliate of Elsevier Inc. All rights reserved. No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, recording, or any information storage and retrieval system, without permission in writing from the publisher. Details on how to seek permission, further information about the Publisher’s permissions policies and our arrangements with organizations such as the Copyright Clearance Center and the Copyright Licensing Agency, can be found at our website: www.elsevier.com/permissions. This book and the individual contributions contained in it are protected under copyright by the Publisher (other than as may be noted herein). Notices Knowledge and best practice in this field are constantly changing. As new research and experience broaden our understanding, changes in research methods, professional practices, or medical treatment may become necessary. Practitioners and researchers must always rely on their own experience and knowledge in evaluating and using any information, methods, compounds, or experiments described herein. In using such information or methods they should be mindful of their own safety and the safety of others, including parties for whom they have a professional responsibility. With respect to any drug or pharmaceutical products identified, readers are advised to check the most current information provided (i) on procedures featured or (ii) by the manufacturer of each product to be administered, to verify the recommended dose or formula, the method and duration of administration, and contraindications. It is the responsibility of practitioners, relying on their own experience and knowledge of their patients, to make diagnoses, to determine dosages and the best treatment for each individual patient, and to take all appropriate safety precautions. To the fullest extent of the law, neither the Publisher nor the authors, contributors, or editors assume any liability for any injury and/or damage to persons or property as a matter of products liability, negligence or otherwise, or from any use or operation of any methods, products, instructions, or ideas contained in the material herein. International Standard Book Number: 978-0-323-08663-9 Publishing Director: Andrew Allen Content Manager: Ellen Wurm-Cutter Publishing Services Manager: Catherine Jackson Senior Project Manager: David Stein Design Direction: Maggie Reid Working together to grow libraries in developing countries Printed in the United States www.elsevier.com | www.bookaid.org | www.sabre.org Last digit is the print number: 9 8 7 6 5 4 3 2 1 This book is dedicated to my family, Tommy and Sean, for their support. And to all the students and professionals I have worked with throughout my career in immunohematology. KDB This third edition is dedicated in loving memorium to my parents, William and Olga Juda, who encouraged my individuality and desire for continuous learning and to my partner, Jack, for his perpetual belief and support of my professional goals. And as always to all of my former CLS students who energized my personal joy of learning and inspired my desire for excellence in teaching. PRH This page intentionally left blank Reviewers Charlotte Bates, MEd, MT(ASCP) Max P. Marschner, MBA, MT(ASCP)SBB, CHS Instructor Manager, Tissue Typing Lab Medical Laboratory Science Department Florida Hospital Medical Center Armstrong Atlantic State University Orlando, Florida Savannah, Georgia Nicole S. Pekarek, MAT, MT(ASCP) Dorothy A. Bergeron, MS, CLS(NCA) Instructor Associate Professor and Program Director Clinical Laboratory Science Instructor Clinical Laboratory Science Program Winston-Salem State University Department of Medical Laboratory Science Winston-Salem, North Carolina University of Massachusetts Dartmouth North Dartmouth, Massachusetts Ellen F. Romani, MHSA, MT(ASCP)DLM, BB Department Chair Kim Boyd, MS, MT(AMT) Medical Laboratory Technology Program Assistant Professor Spartanburg Community College Medical Laboratory Technology Program Spartanburg, South Carolina Amarillo College Amarillo, Texas Judith A Seidel, MT(ASCP)SBB Clinical Instructor, Immunohematology Cara Calvo, MS, MT(ASCP)SH Clinical Laboratory Science Program Program Director and Lecturer Indiana University Health Medical Technology Program Indianapolis, Indiana Department of Laboratory Medicine University of Washington Melissa Volny, MT(ASCP)SBB, MBA Seattle, Washington Coordinator of Transfusion Services Centegra Health System Linda Collins, MS, MT(ASCP) McHenry, Illinois; Instructor Elgin Community College Delaware Technical and Community College Elgin, Illinois Georgetown, Delaware Terry Kotrla, MS, MT(ASCP)BB Department Chair and Professor Medical Laboratory Technology Program Austin Community College Austin, Texas vii This page intentionally left blank Preface Basic & Applied Concepts of Blood Banking and Trans- Chapter summaries, in varying formats, to provide a fusion Practices was developed for students in 2- or succinct overview of the chapter’s important points 4-year medical laboratory science programs, laboratory Critical thinking exercises to illustrate the practical professionals undergoing retraining, and other health applications to the clinical environment care professionals who desire knowledge in routine Illustrations and tables designed to reinforce and sum- blood banking practices. Basic didactic concepts are marize the most important information found in the introduced, and the practical application of these theo- chapter ries to modern transfusion and blood bank settings is The third edition’s presentation of topics was reorga- emphasized. nized to improve the overall flow of the information. We The third edition includes updates to the ever-chang- also included additional details on some topics more ing field of blood banking. Donor criteria and testing appropriate for the 4-year medical laboratory science have been updated to include the current donor restric- programs. tions, infectious disease testing methods, and current The third edition also has an accompanying Evolve requirements for viral marker testing. A new chapter was website where the ancillaries are highlighted. For stu- added to address automation for the transfusion service. dents, the ancillaries include additional case studies and The section on molecular techniques applying to blood access to the laboratory manual. The instructor ancillar- banking was expanded, accompanied by an expanded ies include an image collection that features figures found section on HLA. The chapter on blood components and in the text, an extensive collection of test bank questions therapy includes a description of new products such as as well as answers to the critical thinking exercises, and leukoreduced components and red cell apheresis. PowerPoint presentations for each chapter that include This textbook provides important features to assist illustrations appearing in this text. both the student and the instructor. Each chapter We are very appreciative of the editors at Elsevier for features: their patience and professionalism in the manuscript Chapter outlines listing the important elements in the review and publication process for this third edition. We chapter are proud of the final product, which is user friendly to Learning objectives for use by both the student and students and instructors. the instructor Study questions for self-assessment Kathy D. Blaney Key words with definitions on the same page Paula R. Howard ix This page intentionally left blank Contents PART I: FOUNDATIONS: BASIC SCIENCES AND Inheritance and Nomenclature of HLA, 19 REAGENTS Testing and Identification of HLA and Antibodies, 21 Chapter 1 IMMUNOLOGY: Basic Principles and Hematopoietic Progenitor Cell Transplants, 22 Applications in the Blood Bank, 1 Graft-versus-Host Disease, 23 Platelet Antigens, 23 SECTION 1 CHARACTERISTICS ASSOCIATED WITH ANTIGEN-ANTIBODY REACTIONS, 2 General Properties of Antigens, 2 Chapter 2 BLOOD BANKING REAGENTS: Overview General Properties of Antibodies, 3 and Applications, 28 Molecular Structure, 3 Fab and Fc Regions, 5 SECTION 1 INTRODUCTION TO ROUTINE TESTING IN Comparison of IgM and IgG Antibodies, 5 IMMUNOHEMATOLOGY, 29 IgM Antibodies, 6 Sources of Antigen for Testing, 29 IgG Antibodies, 7 Sources of Antibody for Testing, 30 Primary and Secondary Immune Response, 7 Routine Testing Procedures in the Antigen-Antibody Reactions, 8 Immunohematology Laboratory, 30 Properties That Influence Binding, 8 SECTION 2 INTRODUCTION TO BLOOD BANKING SECTION 2 CHARACTERISTICS ASSOCIATED WITH RED REAGENTS, 31 CELL ANTIGEN-ANTIBODY REACTIONS, 10 Regulation of Reagent Manufacture, 31 Red Cell Antigens, 10 Reagent Quality Control, 32 Red Cell Antibodies, 12 Immunohematology: Antigen-Antibody Reactions SECTION 3 COMMERCIAL ANTIBODY REAGENTS, 32 In Vivo, 12 Polyclonal versus Monoclonal Antibody Transfusion, Pregnancy, and the Immune Products, 32 Response, 12 Polyclonal Antibody Reagents, 33 Complement Proteins, 12 Monoclonal Antibody Reagents, 33 Clearance of Antigen-Antibody Complexes, 14 Monoclonal and Polyclonal Antibody Immunohematology: Antigen-Antibody Reactions Reagents, 34 In Vitro, 14 Reagents for ABO Antigen Typing, 34 Overview of Agglutination, 14 Reagents for D Antigen Typing, 36 Sensitization Stage or Antibody Binding to Low-Protein Reagent Control, 37 Red Cells, 14 Factors Influencing First Stage of SECTION 4 REAGENT RED CELLS, 38 Agglutination, 15 A1 and B Red Cells for ABO Serum Testing, 38 Lattice-Formation Stage or Cell-Cell Screening Cells, 39 Interactions, 16 Antibody Identification Panel Cells, 40 Factors Influencing Second Stage of Agglutination, 16 SECTION 5 ANTIGLOBULIN TEST AND REAGENTS, 40 Grading Agglutination Reactions, 17 Principles of Antiglobulin Test, 40 Hemolysis as an Indicator of Antigen-Antibody Direct Antiglobulin Test, 42 Reactions, 18 Indirect Antiglobulin Test, 43 Sources of Error in Antiglobulin Testing, 43 SECTION 3 HUMAN LEUKOCYTE ANTIGEN (HLA) SYSTEM Antiglobulin Reagents, 44 AND PLATELET IMMUNOLOGY, 19 Polyspecific Antihuman Globulin Reagents, 45 Human Leukocyte Antigens, 19 Monospecific Antihuman Globulin Testing Applications in the Clinical Reagents, 45 Laboratory, 19 IgG-Sensitized Red Cells, 46 xi xii CONTENTS SECTION 6 PRINCIPLES OF ANTIBODY POTENTIATORS AND SECTION 2 ABO AND H BLOOD GROUP SYSTEM LECTINS, 47 ANTIGENS, 80 Low-Ionic-Strength Saline (LISS), 47 General Characteristics of ABO Antigens, 80 Polyethylene Glycol, 48 Inheritance and Development of A, B, and Enzymes, 48 H Antigens, 81 Bovine Serum Albumin, 48 Common Structure for A, B, and H Antigens, 82 Lectins, 49 Development of H Antigen, 82 Development of A and B Antigens, 82 SECTION 7 OTHER METHODS OF DETECTING ANTIGEN- ABO Subgroups, 84 ANTIBODY REACTIONS, 49 Comparison of A1 and A2 Phenotypes, 84 Gel Technology Method, 49 Additional Subgroups of A and B, 85 Microplate Testing Methods, 50 Importance of Subgroup Identification in Donor Solid-Phase Red Cell Adherence Methods, 52 Testing, 86 SECTION 3 GENETIC FEATURES OF ABO BLOOD GROUP Chapter 3 Genetic Principles in Blood SYSTEM, 86 Banking, 59 SECTION 4 ABO BLOOD GROUP SYSTEM ANTIBODIES, 88 SECTION 1 BLOOD GROUP GENETICS, 60 General Characteristics of Human Anti-A and Genetic Terminology, 60 Anti-B, 88 Phenotype versus Genotype, 61 Immunoglobulin Class, 88 Punnett Square, 61 Hemolytic Properties and Clinical Genes, Alleles, and Polymorphism, 61 Significance, 88 Inheritance Patterns, 62 In Vitro Serologic Reactions, 89 Silent Genes, 63 Human Anti-A,B from Group O Individuals, 89 Mendelian Principles, 63 Anti-A1, 89 Chromosomal Assignment, 64 Heterozygosity and Homozygosity, 64 SECTION 5 ABO BLOOD GROUP SYSTEM AND Genetic Interaction, 65 TRANSFUSION, 89 Linkage and Haplotypes, 65 Routine ABO Phenotyping, 89 Crossing Over, 66 Selection of ABO-Compatible Red Blood Cells and Plasma Products for Transfusion, 90 SECTION 2 POPULATION GENETICS, 67 Combined Phenotype Calculations, 67 SECTION 6 RECOGNITION AND RESOLUTION OF ABO Gene Frequencies, 68 DISCREPANCIES, 91 Relationship Testing, 68 Technical Considerations in ABO Phenotyping, 91 Sample-Related ABO Discrepancies, 92 SECTION 3 MOLECULAR GENETICS, 69 ABO Discrepancies Associated with Red Cell Application of Molecular Genetics to Blood Testing, 92 Banking, 69 ABO Discrepancies Associated with Serum or Polymerase Chain Reaction, 70 Plasma Testing, 96 Polymerase Chain Reaction–Based Human Leukocyte Antigen Typing SECTION 7 SPECIAL TOPICS RELATED TO ABO AND H Procedures, 70 BLOOD GROUP SYSTEMS, 100 Molecular Testing Applications in Red Cell Classic Bombay Phenotype, 100 Typing, 72 Secretor Status, 101 Polymerase Chain Reaction–Based Red Cell Typing Procedures, 72 Chapter 5 Rh Blood Group System, 107 SECTION 1 HISTORICAL OVERVIEW OF THE DISCOVERY OF PART II: OVERVIEW OF THE MAJOR BLOOD THE D ANTIGEN, 108 GROUPS SECTION 2 GENETICS, BIOCHEMISTRY, AND Chapter 4 ABO and H Blood Group Systems and TERMINOLOGY, 108 Secretor Status, 77 Fisher-Race: CDE Terminology, 110 SECTION 1 HISTORICAL OVERVIEW OF ABO BLOOD Wiener: Rh-Hr Terminology, 110 GROUP SYSTEM, 79 Rosenfield: Numeric Terminology, 111  CONTENTS xiii International Society of Blood Transfusion: Genetics of Duffy Blood Group System, 135 Standardized Numeric Terminology, 111 Characteristics of Duffy Antibodies, 135 Determining the Genotype from the Duffy System and Malaria, 136 Phenotype, 111 SECTION 5 KIDD BLOOD GROUP SYSTEM, 136 SECTION 3 ANTIGENS OF THE Rh BLOOD GROUP Characteristics and Biochemistry of Kidd SYSTEM, 112 Antigens, 136 D Antigen, 112 Kidd Antigens Facts, 136 Weak D, 112 Biochemistry of Kidd Antigens, 136 Weak D: Genetic, 115 Genetics of Kidd Blood Group System, 137 Weak D: Position Effect, 115 Characteristics of Kidd Antibodies, 137 Weak D: Partial D, 115 Significance of Testing for Weak D, 116 SECTION 6 LUTHERAN BLOOD GROUP SYSTEM, 138 Other Rh Blood Group System Antigens, 117 Characteristics and Biochemistry of Lutheran Compound Antigens, 117 Antigens, 138 G Antigens, 118 Lutheran Antigens Facts, 138 Unusual Phenotypes, 118 Biochemistry of Lutheran Antigens, 139 D-Deletion Phenotype, 118 Genetics of Lutheran Blood Group System, 139 Rhnull Phenotype, 119 Characteristics of Lutheran Antibodies, 139 Rhmod Phenotype, 119 Anti-Lua, 139 Anti-Lub, 139 SECTION 4 Rh ANTIBODIES, 119 General Characteristics, 119 SECTION 7 LEWIS BLOOD GROUP SYSTEM, 140 Clinical Considerations, 119 Characteristics and Biochemistry of Lewis Transfusion Reactions, 119 Antigens, 140 Hemolytic Disease of the Fetus and Newborn, 120 Lewis Antigens Facts, 140 Biochemistry of Lewis Antigens, 140 SECTION 5 LW BLOOD GROUP SYSTEM, 120 Inheritance of Lewis System Antigens, 141 Relationship to the Rh Blood Group System, 120 Characteristics of Lewis Antibodies, 142 Serologic Characteristics, 142 Chapter 6 Other Blood Group Systems, 126 SECTION 8 I BLOOD GROUP SYSTEM AND i ANTIGEN, 142 SECTION 1 WHY STUDY OTHER BLOOD GROUP I and i Antigens Facts, 143 SYSTEMS? 127 Biochemistry of I and i Antigens, 143 Organization of Chapter, 127 Serologic Characteristics of Autoanti-I, 143 Disease Association, 144 SECTION 2 KELL BLOOD GROUP SYSTEM, 129 Characteristics and Biochemistry of Kell Antigens, SECTION 9 P1PK BLOOD GROUP SYSTEM, GLOBOSIDE 129 BLOOD GROUP SYSTEM, AND GLOBOSIDE BLOOD GROUP Kell Antigens Facts, 129 COLLECTION, 144 Biochemistry of Kell Antigens, 129 P1 Antigen, 144 Immunogenicity of Kell Antigens, 130 P Antigen, 144 K0 or Kellnull Phenotype, 130 P1PK and GLOB Blood Group System Antigens Genetics of Kell Blood Group System, 131 Facts, 144 Characteristics of Kell Antibodies, 131 Biochemistry, 145 P1PK and GLOB Blood Group System SECTION 3 Kx BLOOD GROUP SYSTEM, 132 Antibodies, 146 Kx Antigen and Its Relationship to Kell Blood Anti-P1, 146 Group System, 132 Autoanti-P, 146 McLeod Phenotype, 132 Anti-PP1Pk, 147 McLeod Syndrome, 133 SECTION 10 MNS BLOOD GROUP SYSTEM, 147 SECTION 4 DUFFY BLOOD GROUP SYSTEM, 134 M and N Antigens, 147 Characteristics and Biochemistry of Duffy S and s Antigens, 148 Antigens, 134 Genetics and Biochemistry, 148 Duffy Antigens Facts, 134 GPA: M and N Antigens, 148 Biochemistry of Duffy Antigens, 134 GPB: S, s, and U Antigens, 148 xiv CONTENTS Antibodies of MNS Blood Group System, 148 Standards and Regulations Governing the Anti-M, 149 Crossmatch, 191 Anti-N, 150 Crossmatch Procedures, 191 Anti-S, Anti-s, and Anti-U, 150 Serologic Crossmatch, 192 Computer Crossmatch, 192 Limitations of Crossmatch Testing, 193 SECTION 11 MISCELLANEOUS BLOOD GROUP Problem Solving Incompatible Crossmatches, 194 SYSTEMS, 150 SECTION 2 PRINCIPLES OF COMPATIBILITY TESTING, 194 PART III: ESSENTIALS OF PRETRANSFUSION Overview of Steps in Compatibility Testing, 194 TESTING Recipient Blood Sample, 194 Comparison with Previous Records, 196 Chapter 7 Antibody Detection and Repeat Testing of Donor Blood, 196 Identification, 158 Pretransfusion Testing on Recipient Sample, 197 Tagging, Inspecting, Issuing, and Transfusing SECTION 1 ANTIBODY DETECTION, 159 Blood Products, 198 Antibody Screen, 159 Autocontrol and Direct Antiglobulin Test, 160 SECTION 3 SPECIAL TOPICS, 199 Potentiators, 161 Urgent Requirement for Blood and Blood Patient History, 161 Components, 199 Massive Transfusion, 201 SECTION 2 ANTIBODY IDENTIFICATION, 162 Maximum Surgical Blood Order Schedule, 201 Initial Panel, 162 Type and Screen Protocols, 201 Panel Interpretation: Single Antibody Crossmatching Autologous Blood, 202 Specificity, 163 Crossmatching of Infants Younger than 4 Months Autocontrol, 165 Old, 202 Phases, 165 Pretransfusion Testing for Non–Red Blood Cell Reaction Strength, 165 Products, 203 Ruling Out, 165 Matching the Pattern, 166 Chapter 9 Blood Bank Automation for Transfusion Rule of Three, 166 Services, 208 Patient’s Phenotype, 166 Multiple Antibodies, 166 SECTION 1 INTRODUCTION TO AUTOMATION IN Multiple Antibody Resolution, 168 IMMUNOHEMATOLOGY, 208 Additional Techniques, 168 Forces Driving the Change to Automation, 209 Antibodies to High-Frequency Antigens, 169 Benefits and Barriers of Automated Additional Testing, 170 Instruments, 209 High-Titer, Low-Avidity Antibodies, 170 Potential Benefits, 209 Antibodies to Low-Frequency Antigens, 171 Potential Challenges, 210 Enhancing Weak IgG Antibodies, 171 Characteristics of an Ideal Instrument for the Cold Alloantibodies, 172 Blood Bank, 211 SECTION 3 AUTOANTIBODIES, 174 SECTION 2 SELECTION OF AUTOMATION TO MEET Cold Autoantibodies, 174 LABORATORY NEEDS, 211 Specificity, 175 Vendor Assessment, 211 Avoiding Cold Autoantibody Reactivity, 176 Base Technology Assessment, 212 Adsorption Techniques, 177 Instrument Assessment, 212 Warm Autoantibodies, 177 Specificity, 178 SECTION 3 AUTOMATED TESTING SYSTEMS, 213 Elution, 178 Automated Systems for Solid Phase Red Cell Adsorption, 180 Adherence Assays, 213 Hemagglutination Assays, 214 Chapter 8 Compatibility Testing, 188 Solid Phase Red Cell Adherence Assays, 215 SolidscreenR II Technology, 216 SECTION 1 PRINCIPLES OF THE CROSSMATCH, 190 ErytypeR S Technology, 217 What Is a Crossmatch? 190 Automated System for Gel Technology Purposes of Crossmatch Testing, 191 Assays, 220  CONTENTS xv PART IV: CLINICAL CONSIDERATIONS IN SECTION 3 PREDICTION OF HEMOLYTIC DISEASE OF THE IMMUNOHEMATOLOGY FETUS AND NEWBORN, 250 Maternal History, 250 Chapter 10 Adverse Complications of Antibody Titration, 250 Transfusions, 226 Ultrasound Techniques, 251 SECTION 1 OVERVIEW OF ADVERSE REACTIONS TO Amniocentesis, 252 TRANSFUSION, 227 Cordocentesis, 252 Fetal Genotyping, 253 Hemovigilance Model, 227 Recognition of a Transfusion Reaction, 227 SECTION 4 POSTPARTUM TESTING, 253 SECTION 2 CATEGORIES OF TRANSFUSION Postpartum Testing of Infants and Mothers, 254 REACTIONS, 228 D Testing, 254 ABO Testing, 255 Hemolytic Transfusion Reaction, 228 Direct Antiglobulin Test, 255 Acute Hemolytic Transfusion Reaction, 228 Intrauterine Transfusions, 255 Delayed Hemolytic Reaction, 230 Non–Immune-Mediated Mechanisms of Red Cell SECTION 5 PREVENTION OF HEMOLYTIC DISEASE OF THE Destruction, 231 FETUS AND NEWBORN, 255 Delayed Serologic Transfusion Reactions, 232 Antepartum Administration of Rh Immune Febrile Nonhemolytic Transfusion Reactions, 233 Globulin, 256 Allergic and Anaphylactic Transfusion Reactions, Postpartum Administration of Rh Immune 234 Globulin, 256 Transfusion-Related Acute Lung Injury, 234 Screening for Fetomaternal Hemorrhage, 257 Transfusion-Associated Graft-versus-Host Disease, Quantifying Fetomaternal Hemorrhage, 258 235 Bacterial Contamination of Blood Products, 236 SECTION 6 TREATMENT OF HEMOLYTIC DISEASE OF THE Transfusion-Associated Circulatory Overload, 237 FETUS AND NEWBORN, 258 Transfusion Hemosiderosis, 237 In Utero Treatment, 258 Citrate Toxicity, 237 Postpartum Treatment, 259 Posttransfusion Purpura, 238 Phototherapy, 259 SECTION 3 EVALUATION AND REPORTING A Exchange Transfusion, 259 TRANSFUSION REACTION, 238 Selection of Blood and Compatibility Testing for Exchange Transfusion, 260 Initiating a Transfusion Reaction Investigation, 238 Additional Laboratory Testing in a Transfusion PART V: BLOOD COLLECTION AND TESTING Reaction, 240 Records and Reporting of Transfusion Reactions Chapter 12 Donor Selection and Phlebotomy, 267 and Fatalities, 241 Hemovigilance Component, 241 SECTION 1 DONOR SCREENING, 268 Records, 241 Registration, 268 FDA Reportable Fatalities, 241 Educational Materials, 268 Health History Interview, 270 Questions for Protection of the Donor, 270 Chapter 11 Hemolytic Disease of the Fetus and Questions for Protection of the Recipient, 272 Newborn, 246 Physical Examination, 275 SECTION 1 ETIOLOGY OF HEMOLYTIC DISEASE OF THE General Appearance, 275 FETUS AND NEWBORN, 247 Hemoglobin or Hematocrit Determination, 275 Temperature, 275 SECTION 2 OVERVIEW OF HEMOLYTIC DISEASE OF THE Blood Pressure, 275 FETUS AND NEWBORN, 247 Pulse, 275 Rh Hemolytic Disease of the Fetus and Weight, 275 Newborn, 248 Informed Consent, 276 ABO Hemolytic Disease of the Fetus and Newborn, 249 SECTION 2 PHLEBOTOMY, 276 Alloantibodies Causing Hemolytic Disease of the Identification, 276 Fetus and Newborn Other than Anti-D, 249 Bag Labeling, 276 xvi CONTENTS Arm Preparation and Venipuncture, 277 PART VI: BLOOD COMPONENT PREPARATION AND Adverse Donor Reactions, 277 TRANSFUSION THERAPY Postdonation Instructions and Care, 277 Chapter 14 Blood Component Preparation and SECTION 3 SPECIAL BLOOD COLLECTION, 279 Therapy, 304 Autologous Donations, 279 SECTION 1 BLOOD COLLECTION AND STORAGE, 305 Preoperative Collection, 279 Normovolemic Hemodilution, 280 Storage Lesion, 306 Blood Recovery, 280 Types of Anticoagulant-Preservative Directed Donations, 280 Solutions, 307 Apheresis, 281 Additive Solutions, 307 Therapeutic Phlebotomy, 281 Rejuvenation Solution, 308 SECTION 2 BLOOD COMPONENT PREPARATION, 309 Chapter 13 Testing of Donor Blood, 286 Whole Blood, 311 SECTION 1 OVERVIEW OF DONOR BLOOD TESTING, 286 Indications for Use, 311 Required Testing on Allogeneic and Autologous Red Blood Cell Components, 311 Donor Blood, 287 Indications for Use, 311 Red Blood Cells Leukocytes Reduced, 312 SECTION 2 IMMUNOHEMATOLOGIC TESTING OF DONOR Apheresis Red Blood Cells, 313 UNITS, 287 Frozen Red Blood Cells, 314 ABO and D Phenotype, 287 Deglycerolized Red Blood Cells, 314 Antibody Screen, 288 Washed Red Blood Cells, 315 Red Blood Cells Irradiated, 315 SECTION 3 INFECTIOUS DISEASE TESTING OF DONOR Platelet Components, 316 UNITS, 288 Indications for Use, 316 Serologic Tests for Syphilis, 288 Platelets, 317 Rapid Plasma Reagin Test, 288 Pooled Platelets, 317 Hemagglutination Test for Treponema pallidum Apheresis Platelets, 317 Antibodies, 289 Platelets Leukocytes Reduced, 318 Confirmatory Testing for Syphilis, 289 Plasma Components, 318 Principles of Viral Marker Testing, 289 Fresh Frozen Plasma, Plasma Frozen within Enzyme-Linked Immunosorbent Assay, 289 24 Hours of Phlebotomy, 318 Nucleic Acid Testing, 291 Cryoprecipitated Antihemophilic Chemiluminescence, 291 Factor, 319 Controls, 291 Apheresis Granulocytes, 321 Sensitivity and Specificity, 292 SECTION 3 DISTRIBUTION AND ADMINISTRATION, 321 Viral Hepatitis, 292 Hepatitis Viruses, 292 Labeling, 321 Hepatitis Tests, 294 Storage and Transportation, 323 Human Retroviruses, 295 Transportation of Blood Components, 323 Human Immunodeficiency Virus Types 1 Administration of Blood Components, 324 and 2, 296 Nucleic Acid Testing for Ribonucleic Chapter 15 Transfusion Therapy in Selected Acid of Human Immunodeficiency Virus Patients, 329 Type 1, 297 Human T-Lymphotropic Virus Types I SECTION 1 TRANSFUSION PRACTICES, 329 and II, 297 Urgent and Massive Transfusion, 329 Western Blotting, 297 Cardiac Surgery, 330 West Nile Virus, 298 Neonatal and Pediatric Transfusion Issues, 331 Recipient Tracing (Look-Back), 299 Transplantation, 332 Additional Tests Performed on Donor Organ Transplants, 333 Blood, 299 Hematopoietic Progenitor Cell Transplantation, Cytomegalovirus, 299 333 Chagas Disease, 299 Therapeutic Apheresis, 335 Testing for Bacterial Contamination of Blood Oncology, 336 Components, 300 Chronic Renal Disease, 337  CONTENTS xvii Hemolytic Uremic Syndrome and Thrombotic Change Control, 352 Thrombocytopenic Purpura, 338 Personnel Qualifications, 352 Anemias Requiring Transfusion Support, 339 Supplier Qualification, 354 Sickle Cell Anemia, 339 Error Management, 354 Thalassemia, 339 Validation, 355 Immune Hemolytic Anemias, 340 Facilities and Equipment, 355 Hemostatic Disorders, 340 Proficiency Testing, 355 Label Control, 356 SECTION 2 ALTERNATIVES TO TRANSFUSION, 341 SECTION 3 SAFETY, 356 Standard versus Universal Precautions, 356 PART VII: QUALITY AND SAFETY ISSUES Blood Bank Safety Program, 356 Physical Space, Safety Equipment, Protective Chapter 16 Quality Assurance and Regulation of Devices, and Warning Signs, 357 the Blood Industry and Safety Issues in Decontamination, 359 the Blood Bank, 345 Chemical Storage and Hazards, 359 SECTION 1 REGULATORY AND ACCREDITING AGENCIES Radiation Safety, 359 FOR QUALITY AND SAFETY, 346 Biohazardous Wastes, 359 Food and Drug Administration, 346 Storage and Transportation of Blood and Blood AABB, 347 Components, 360 Other Safety Regulations, 347 Personal Injury and Reporting, 360 Occupational Safety and Health Act, 347 Employee Education, 360 Environmental Protection Agency, 347 SECTION 2 QUALITY ASSURANCE AND GOOD APPENDIX A: ANSWERS TO STUDY MANUFACTURING PRACTICES, 348 QUESTIONS, 365 Quality Assurance, 348 Quality Assurance Department, 348 Good Manufacturing Practices, 348 GLOSSARY, 367 Components of a Quality Assurance Program, 348 Records and Documents, 348 Standard Operating Procedures, 351 INDEX, 373 This page intentionally left blank FOUNDATIONS: BASIC SCIENCES AND REAGENTS PART I 1 IMMUNOLOGY: Basic Principles and Applications in the Blood Bank CHAPTER OUTLINE SECTION 1: CHARACTERISTICS ASSOCIATED WITH Immunohematology: Antigen-Antibody Reactions ANTIGEN-ANTIBODY REACTIONS In Vitro General Properties of Antigens Overview of Agglutination General Properties of Antibodies Sensitization Stage or Antibody Binding to Red Cells Molecular Structure Factors Influencing First Stage of Agglutination Fab and Fc Regions Lattice-Formation Stage or Cell-Cell Interactions Comparison of IgM and IgG Antibodies Factors Influencing Second Stage of IgM Antibodies Agglutination IgG Antibodies Grading Agglutination Reactions Primary and Secondary Immune Response Hemolysis as an Indicator of Antigen-Antibody Antigen-Antibody Reactions Reactions Properties That Influence Binding SECTION 3: HUMAN LEUKOCYTE ANTIGEN (HLA) SECTION 2: CHARACTERISTICS ASSOCIATED WITH RED SYSTEM AND PLATELET IMMUNOLOGY CELL ANTIGEN-ANTIBODY REACTIONS Human Leukocyte Antigens Red Cell Antigens Testing Applications in the Clinical Laboratory Red Cell Antibodies Inheritance and Nomenclature of HLA Immunohematology: Antigen-Antibody Reactions Testing and Identification of HLA and In Vivo Antibodies Transfusion, Pregnancy, and the Immune Response Hematopoietic Progenitor Cell Transplants Complement Proteins Graft-versus-Host Disease Clearance of Antigen-Antibody Complexes Platelet Antigens LEARNING OBJECTIVES On completion of this chapter, the reader should be able to: 1. Define the following terms in relation to red cells and 7. List the variables in the agglutination test that affect transfusion: antigen, immunogen, epitopes, and antigenic sensitization and lattice formation. determinants. 8. Accurately grade and interpret observed agglutination 2. Describe the characteristics of antigens that are located reactions using the agglutination grading scale for on red cells, white cells, and platelets. antigen-antibody reactions performed in test tubes. 3. Diagram the basic structure of an IgG molecule 9. Compare the classical and alternative pathways of and label the following components: heavy and light complement activation. chains, Fab, and Fc regions, variable region, hinge 10. Outline the biological effects mediated by complement region, antigen-binding site, and macrophage-binding proteins in the clearance of red cells. site. 11. Recognize hemolysis in an agglutination reaction and 4. Compare and contrast IgM and IgG antibodies with explain the significance. regard to structure, function, and detection by 12. Outline how the immune system responds to antigen agglutination reactions. stimulation through transfusion and pregnancy. Explain 5. Distinguish the primary and secondary immune response the factors that cause variations in these in vivo with regard to immunoglobulin class, immune cells responses. involved, level of response, response time, and antibody 13. Using the principles of tissue matching, select the best affinity. potential graft given the HLA typing and antibody 6. Apply the properties that influence the binding of an specificities. antigen and antibody to agglutination tests to achieve 14. Predict the probable HLA typing results in a family optimal results. study performed for graft selection. 1 2 PART I n Foundations: Basic Sciences and Reagents 15. Compare and contrast the class I and II MHC complexes 17. Define graft-versus-host disease (GVHD) and select with regard to antigens, their associated immune cells, methods of prevention in transfusion and and their role in immunity. transplantation. 16. Explain the role of HLA testing in platelet transfusion 18. Outline the serologic test methods used in HLA typing support, organ transplants, and hematopoietic progenitor and antibody identification. cell transplants. Immunohematology: study The science of immunohematology embodies the study of blood group antigens and of blood group antigens and antibodies. Immunohematology is closely related to the field of immunology because it antibodies. involves the immune response to the transfusion of cellular elements. Red cells (erythro- cytes), white cells (leukocytes), and platelets are cellular components that can potentially initiate immune responses after transfusion. To enhance the reader’s understanding of the physiology involved in this immune response, this text begins with an overview of the immune system with an emphasis on the clinical and serologic nature of antibodies and antigens. SECTION 1 CHARACTERISTICS ASSOCIATED WITH ANTIGEN-ANTIBODY REACTIONS Antigen: foreign molecules that bind specifically to an antibody or GENERAL PROPERTIES OF ANTIGENS a T-cell receptor. An antigen is a molecule that binds to an antibody or T-cell receptor. This binding can Allogeneic: cells or tissue from a occur within the body (in vivo) or in a laboratory test (in vitro). In chemical terms, genetically different individual. antigens are large-molecular-weight proteins (including conjugated proteins such as gly- Autologous: cells or tissue from coproteins, lipoproteins, and nucleoproteins) and polysaccharides (including lipopolysac- self. charides). These protein and polysaccharide antigens may be located on the surfaces of cell membranes or may be an integral portion of the cell membrane. Antigens are located Hapten: small-molecular-weight on viruses, bacteria, fungi, protozoa, blood cells, organs, and tissues. particle that requires a carrier molecule to be recognized by the Transfused red cells contain antigens that may be recognized as foreign to the indi- immune system. vidual receiving the blood. These antigens are called allogeneic because they are unfamil- iar to the individual being transfused but are derived from the same species. These foreign B lymphocytes (B cells): antigens may elicit an immune response in the recipient. The body’s immune system lymphocytes that mature in the normally recognizes and tolerates self-antigens. These antigens are termed autologous bone marrow, differentiate into plasma cells when stimulated because they originate from the individual. However, the failure to tolerate self-antigens by an antigen, and produce may cause an immune response against cells or tissue from self. This immune response antibodies. to self may result in various forms of autoimmune disease. In terms of transfusion, an allogeneic transfusion involves the exposure to antigens that are different from the indi- T lymphocytes (T cells): vidual receiving a transfusion, whereas an autologous transfusion involves antigens that lymphocytes that mature in the thymus and produce cytokines originated in the recipient. to activate the immune cells The concept of an antigen having sufficient size to induce an immune response con- including the B cell. trasts with a hapten, which is a small-molecular-weight particle that requires a carrier molecule to initiate the immune response. Haptens may include medications such as Cytokines: secreted proteins that penicillin and are sometimes referred to as partial antigens. regulate the activity of other cells by binding to specific receptors. The immune response to foreign or potentially pathogenic antigens involves a complex They can increase or decrease cell interaction between several types of leukocytes. In the transfusion setting, immune proliferation, antibody production, response is primarily humoral, involving mainly B lymphocytes (B cells). Following a and inflammation reactions. transfusion, the recipient’s B cells may “recognize” these foreign red cell antigens through B-cell receptors (Fig. 1-1). This recognition causes the B cells to present the antigen to Memory B cells: B cells produced after the first exposure the T lymphocytes (T cells). After presentation, the T-cell cytokines signal the B cells to that remain in the circulation and be transformed into plasma cells that produce antibodies with the same specificity as the can recognize and respond to an original B-cell receptors. These antibodies are glycoprotein molecules that continue to antigen faster. circulate and specifically recognize and bind to the foreign antigen that originally created the response. Memory B cells are also made at this time. If there is a reexposure at a later Plasma cells: antibody- producing B cells that have date, the memory B cells can respond quickly and change into antibody-producing plasma reached the end of their cells; memory B cells do not require presentation to the T cell to be activated. Memory differentiating pathway. B cells allow a fast response to an antigen, an important principle used in vaccination.  CHAPTER 1 n Immunology: Basic Principles and Applications in the Blood Bank 3 Lymphocyte Mature precursor lymphocyte Lymphocyte clones with diverse receptors Antigens that exhibit the arise in generative greatest degree of foreignness lymphoid organs from the host elicit the strongest immune response. Clones of mature lymphocytes specific for many antigens enter lymphoid tissues Antigen X Antigen Y Antigen-specific clones are activated ("selected") by antigens Plasma cells produce antibodies specific for the antigen Anti-X Anti-Y antibody antibody Fig. 1-1 B-cell response to an antigen. Mature lymphocytes develop receptors for antigens before they encounter the antigen. The antigen stimulates the lymphocyte that has the receptor with the best fit. These lymphocytes are signaled to produce a B-cell clone, which differentiates into plasma cells that produce an antibody with a single specificity. (From Abbas AK, Lichtman AH: Basic immunology, ed 3, Philadelphia, 2011, Saunders.) Antigenic determinants: sites Many different antibodies to foreign antigens can be produced in the immune response, on an antigen that are recognized each binding to a different antigen on the surface. For example, red cells have many dif- and bound by a particular antibody or T-cell receptor (also ferent antigens on their surface. When red cells from one donor are transfused to a patient, called epitopes). several different antibodies may be produced in the immune response to the transfused red cells. The different antigenic determinants, also called epitopes, on a red cell can elicit Epitopes: single antigenic the production of different antibodies. Each B cell has a unique specificity, which is determinants; functionally, they “selected” by the antigenic determinant to expand into a clone of identical plasma cells are the parts of the antigen that combine with the antibody. making antibodies with the same specificity as the original B-cell receptor. The term antigen is often inappropriately used as a synonym for an immunogen. An Clone: family of cells or immunogen is an antigen that is capable of eliciting an immune response in the body. organisms having genetically The immune system’s ability to recognize an antigen and respond to it varies among identical constitution. individuals and can even vary within an individual at a given time. Several important Immunogen: antigen in its role characteristics of a molecule contribute to its degree of immunogenicity (Table 1-1). For of eliciting an immune response. example, different biological materials have varying degrees of immunogenicity. Protein molecules are the most immunogenic, followed by carbohydrates and lipids, which tend Carbohydrates: simple sugars, to be immunologically inert. In addition, complex compounds, such as a protein- such as monosaccharides and carbohydrate combination, are more immunogenic than simpler molecules. Antigens on starches (polysaccharides). red cells, white cells, and platelets vary in their ability to elicit an immune response. Lipids: fatty acids and glycerol compounds. GENERAL PROPERTIES OF ANTIBODIES An immunogen is an antigen Molecular Structure that provokes the immune Antibody molecules are glycoproteins composed of four polypeptide chains joined together response. Not all antigens are by disulfide bonds (Fig. 1-2). The terms antibody and immunoglobulin (Ig) are often used immunogens. 4 PART I n Foundations: Basic Sciences and Reagents Secreted IgG Light Antigen- Heavy chain binding site chain N N N VL N S S S S S S VH S S CL S S S S S S CH1 S S CC Fab Hinge region Fc receptor/ S S complement S C 2 S H binding sites Fc region S S S SCH3 Tail piece C C Disulfide bond SS Ig domain S S Fig. 1-2 Basic structure of an IgG molecule. Antigen binds to the variable region of the heavy and light chains. The variable region (VL and VH) is part of Fab (fragment, antigen-binding). The opposite end, composed of the heavy chain, is constant for each type of immunoglobulin. It is called the Fc (fragment, crystallizable) region, which determines the antibody function. The Fc region contains the complement binding region and the cell activation region. (Modified from Abbas AK, Lichtman AH: Basic immunology, ed 3, Philadelphia, 2011, Saunders.) Factors Contributing to Immunogenicity: TABLE 1-1 Properties of the Antigen Antibody: glycoprotein Chemical composition and Proteins are the best immunogens, followed by complex (immunoglobulin) that recognizes complexity of the antigen carbohydrates a particular epitope on an antigen Degree of foreignness Immunogen must be identified as nonself; the greater the and facilitates clearance of that difference from self, the greater likelihood of eliciting antigen. an immune response Immunoglobulin: antibody; Size Molecules with a molecular weight >10,000 D are better glycoprotein secreted by plasma immunogens cells that binds to specific Dosage and antigen density Number of red cells introduced and the amount of epitopes on antigenic substances. antigen that they carry contribute to the likelihood of Heavy chains: larger an immune response polypeptides of an antibody Route of administration Manner in which the antigenic stimulus is introduced; molecule composed of a variable intramuscular or intravenous injections are generally and constant region; five major better routes for eliciting an immune response classes of heavy chains determine the isotype of an antibody. Light chains: smaller synonymously. Five classifications of antibodies are designated as IgG, IgA, IgM, IgD, polypeptides of an antibody and IgE. The five classes are differentiated on the basis of certain physical, chemical, and molecule composed of a variable and constant region; two major biological characteristics. Each antibody molecule has two identical heavy chains and two types of light chains exist in identical light chains joined by disulfide bond (S-S) bridges. These molecular bridges humans (kappa and lambda). provide flexibility to the molecule to change its three-dimensional shape.  CHAPTER 1 n Immunology: Basic Principles and Applications in the Blood Bank 5 Variable region is specific for different epitopes Each immunoglobulin Red cell with various epitopes, molecule consists of two represented by shapes identical heavy chains and Fig. 1-3 Variable region of an immunoglobulin. The specificity of an antibody is determined by the unique two identical light chains variable region that “fits” antigenic determinants or epitopes. (either kappa or lambda). The five distinctive heavy-chain molecules distinguish the class or isotype. Each heavy Isotype: one of five types of chain imparts characteristic features, which permit them to have unique biological func- immunoglobulins determined by the heavy chain: IgM, IgG, IgA, tions. For example, the IgA family, which possesses alpha heavy chains, is the only anti- IgE, and IgD. body class capable of residing in mucosal linings. IgE antibodies can activate mast cells causing immediate hypersensitivity reactions. IgD is an antigen receptor on the naive B Kappa chains: one of the two cell.1 The immunoglobulins most involved in transfusion medicine are IgM and IgG, and types of light chains that make up these are discussed in more detail subsequently. There are two types of light chains: kappa an immunoglobulin. chains and lambda chains. Antibodies possess either two kappa or two lambda chains Lambda chains: one of the two but never one of each. types of light chains that make up Each heavy-chain and light-chain molecule also contains variable regions and constant an immunoglobulin. regions (or domains). The constant regions of the heavy-chain domain impart the unique antibody class functions, such as the activation of complement or the attachment to Variable regions: amino- terminal portions of certain cells. The variable regions of both the heavy chains and the light chains are con- immunoglobulins and T-cell cerned with antigen binding and constitute the area of the antibody that contains idiotope receptor chains that are highly (the idiotypic portion). This area is the binding site or pocket into which the antigen fits variable and responsible for the (Fig. 1-3). T-cell receptors also have specific antigen-binding receptors referred to as the antigenic specificity of these idiotope. The hinge region of the antibody molecule imparts flexibility to the molecule molecules. for combination with the antigen. Constant regions: nonvariable portions of the heavy and light Fab and Fc Regions chains of an immunoglobulin. Early experiments to identify antibody structure and function used enzymes to cleave the immunoglobulin molecule. Enzymes such as pepsin and papain can divide the immuno- Idiotope: variable part of an antibody or T-cell receptor; the globulin molecule to produce two fragments known as Fab (fragment, antigen-binding) antigen-binding site. and Fc (fragment, crystallizable). Fab contains the portion of the molecule that binds to the antigenic determinant. Fc consists of the remainder of the constant domains of the Hinge region: portion of the two heavy chains linked by disulfide bonds (see Fig. 1-2). Certain immune cells, such as immunoglobulin heavy chains macrophages and neutrophils, possess receptors for the Fc region of an immunoglobulin. between the Fc and Fab region; provides flexibility to the molecule These immune cells are able to bind the Fc portion of antibodies that are attached to red to allow two antigen-binding sites cells or pathogens and assist in their removal by phagocytosis. This mechanism is one to function independently. way that antibodies facilitate the removal of potential harmful antigens (Fig. 1-4). In transfusion medicine, the antibodies attached to red cell antigens can signal clearance in Extravascular hemolysis: red the liver and spleen, a process called extravascular hemolysis. cell destruction by phagocytes residing in the liver and spleen usually facilitated by IgG COMPARISON OF IgM AND IgG ANTIBODIES opsonization. Because IgM and IgG antibodies have the most significance in immunohematology, the following discussion focuses on these two immunoglobulins. Table 1-2 summarizes important features of IgM and IgG antibodies. 6 PART I n Foundations: Basic Sciences and Reagents Fc receptor Breakdown Opsonization Binding of signals and removal IgG to Fc receptors Phagocytosis of RBC activation of of RBC of RBC in the by IgG on phagocyte phagocyte liver and spleen RBC RBC IgG antibody Signals Phagocyte Fc receptor Fig. 1-4 Antibody attaches to the Fc receptor on a macrophage to signal clearance. The variable portion of the immunoglobulin attaches to the antigen on the red cell, while the macrophage attaches to the Fc portion. The red cell is transported to the spleen and liver for clearance. (Modified from Abbas AK, Lichtman AH: Basic immunology, ed 3, Philadelphia, 2011, Saunders.) TABLE 1-2 Comparison of IgM and IgG CHARACTERISTIC IgM IgG Heavy-chain composition Mu (µ) Gamma (γ) Light-chain composition Kappa (κ) or lambda (λ) Kappa (κ) or lambda (λ) J chain Yes No Molecular weight (D) 900,000 150,000 Valence 10 2 Total serum concentration (%) 10 70-75 Serum half-life (days) 5 23 Crosses the placenta No Yes Activation of classical Yes; very efficient Yes; not as efficient pathway of complement Clearance of red cells Intravascular Extravascular Detection in laboratory tests Immediate-spin Antiglobulin test From Abbas AK, Lichtman AH: Basic immunology, ed 3, Philadelphia, 2011, Saunders. The visible agglutination of IgM Antibodies antigen-positive red cells with When the B cells initially respond to a foreign antigen, they produce IgM antibodies first. IgM antibodies in vitro is also The IgM molecule consists of five basic immunoglobulin units containing two mu heavy referred to as immediate-spin or direct agglutination. chains and two light chains held together by a joining chain (J chain) (Fig. 1-5). Classified as a large pentamer structurally, one IgM molecule contains 10 potential antigen-combining Valency: number of epitopes per sites or has a valency of 10. Because of their large structure and high valency, these mol- molecule of antigen. ecules are capable of visible agglutination of antigen-positive red cells suspended in saline. The agglutination of red cell antigens by IgM antibodies is also referred to as immediate- spin and direct agglutination. IgM antibodies constitute about 10% of the total serum immunoglobulin concentration.1 An important functional feature associated with IgM antibodies is the ability to acti- vate the classical pathway of complement with great efficiency. Only one IgM molecule  CHAPTER 1 n Immunology: Basic Principles and Applications in the Blood Bank 7 Antigen-binding sites Heavy chain Kappa or lambda light chain J chain (joining chain) Basic immunoglobulin unit Fig. 1-5 Pentamer structure of the IgM molecule. Five basic immunoglobulin units exist with 10 antigen- binding sites. is required for the initiation of the classical pathway in complement activation. Complete activation of the classical pathway of complement results in hemolysis of the red cells and intravascular destruction (intravascular hemolysis). Antibodies of the ABO blood Intravascular hemolysis: red group system are typically IgM and can cause rapid hemolysis of red cells if an incompat- cell lyses occurring within the ible unit of blood is transfused. The complement system is discussed later in this chapter. blood vessels usually by IgM activation of complement. IgG Antibodies The IgG antibody molecule consists of a four-chain unit with two gamma heavy chains and two light chains, either kappa or lambda in structure. This form of an immunoglobu- lin molecule is known as a monomer. IgG antibodies constitute about 70% to 75% of the total immunoglobulin concentration in serum.1 The molecule is bivalent; it possesses Bivalent: having a combining two antigen-combining sites. Because of the relatively small size and bivalent structure of power of two. the molecule, most IgG antibodies are not effective in producing a visible agglutinate with Hemolytic disease of the antigen-positive red cells suspended in saline. The antigen-antibody complexes can be fetus and newborn: condition viewed with the use of the antiglobulin test discussed in Chapter 2. caused by destruction of fetal or Fc receptors on placental cells allow the transfer of IgG antibodies across the placenta neonatal red cells by maternal during pregnancy. This transfer of IgG antibodies from the mother to the fetus protects antibodies. newborns from infections. The mother’s IgG antibodies may also cause destruction of fetal red cells in a condition called hemolytic disease of the fetus and newborn (HDFN). The antiglobulin test method This condition occurs if the mother makes an antibody to red cell antigens from exposure is necessary to detect through transfusions or prior pregnancies. If the fetus has the corresponding antigen, the antigen-antibody complexes involving IgG antibodies IgG antibodies target the red cells for destruction. Laboratory testing to detect this process in vitro. is discussed in subsequent chapters. Four IgG subclasses (IgG1, IgG2, IgG3, and IgG4) exist as a result of minor variations in the gamma heavy chains. The amino acid differences in these heavy chains affect the biological activity of the molecule. Subclasses IgG1 and IgG3 are most effective in activat- ing the complement system.2 Because of their immunoglobulin structure, two molecules of IgG are necessary to initiate the classical pathway of complement activation. PRIMARY AND SECONDARY IMMUNE RESPONSE Immunologic response after exposure to an antigen is influenced by the host’s previous Primary immune response: history with the foreign material. There are two types of immune responses: primary and immune response induced by secondary. The primary immune response is elicited on first exposure to the foreign initial exposure to the antigen. 8 PART I n Foundations: Basic Sciences and Reagents antigen. The primary response is characterized by a lag phase of approximately 5 to 10 days and is influenced by the characteristics of the antigen and immune system of the host. Host properties that can contribute to the antigen response include the following: Age Route of administration Genetic makeup Overall health—stress, fatigue, disease Medications (immunosuppressive) Lag phases may extend for longer periods. During this period, no detectable circulating antibody levels exist within the host. After this lag period, antibody concentrations Affinity maturation: process of increase and sustain a plateau before a decline in detectable antibody levels. IgM antibod- somatic mutations in the ies are produced first, followed by the production of IgG antibodies. The specificity of immunoglobulin gene causing the original IgM molecule (determined by the variable region) is the same as the specificity the formation of variations in the of the IgG molecule seen later in the immune response. As a result of a process of gene affinity of the antibody to the rearrangement in the B cell, the affinity of the antibody produced after each exposure antigen. B cells with the highest affinity are “selected” for the best increases. This process is called affinity maturation, and it is the reason why antibodies fit, and the resulting antibody is often produce a stronger reaction in laboratory tests if the patient has had repeated stronger. exposure to the antigen. The second contact with the identical antigen initiates a secondary immune response, Secondary immune response: or anamnestic response, within 1 to 3 days of exposure. Because of the significant pro- immune response induced after a second exposure to the antigen, duction of memory B cells from the initial exposure, the concentrations of circulating which activates the memory antibody are much higher and sustained for a much longer period. Antibody levels are lymphocytes for a quicker many times higher because of the larger number of plasma cells. IgM antibodies are also response. generated in the secondary immune response. However, the principal antibody produced is of the IgG class (Fig. 1-6). In the clinical setting, detecting a higher level of the IgM Anamnestic response: secondary immune response. form of an antibody may indicate an acute or early exposure to a pathogen, whereas finding an increase in the IgG form of an antibody of the same specificity may indicate a chronic or late infection. ANTIGEN-ANTIBODY REACTIONS Multiple stimulations of the Properties That Influence Binding immune system with the same The binding of an antigen and antibody follows the law of mass action and is a reversible antigen produce antibodies process. This union complies with the principles of a chemical reaction that has reached with increased binding equilibrium. When the antigen and antibody combine, an antigen-antibody complex or strength as a result of affinity maturation. immune complex is produced. The amount of antigen-antibody complex formation is determined by the association constant of the reaction. The association constant drives Immune complex: complex of the forward reaction rate, whereas the reverse reaction rate is influenced by the dissocia- one or more antibody molecules tion constant. When the forward reaction rate is faster than the reverse reaction rate, bound to an antigen. antigen-antibody complex formation is favored. A higher association constant influences greater immune complex formation at equilibrium (Fig. 1-7). Several properties influence the binding of antigen and antibody. The goodness of fit and the complementary nature of the antibody for its specific epitope contribute to the strength and rate of the reaction. Factors such as the size, shape, and charge of an antigen determine the binding of the antigen to the complementary antibody. This concept of goodness of fit is most easily seen by viewing the antigen-antibody binding as a lock-and-key fit (Fig. 1-8). If the shape of the antigen is altered, the fit of the antigen for the antibody is changed. Likewise, if the charge of the antigen is altered, the binding properties of the antigen and antibody are affected. The strength of binding Affinity: strength of the binding between a single combining site of an antibody and the epitope of an antigen is called between a single antibody and an the affinity. epitope of an antigen. When the immune complex has been generated, the complex is held together by non- covalent attractive forces, including electrostatic forces (ionic bonding), hydrogen bonding, Avidity: overall strength of hydrophobic bonding, and van der Waals forces. The influence of these forces on immune reaction between several epitopes and antibodies; depends on the complex stability is described further in Table 1-3. The cumulative effect of these forces affinity of the antibody, valency, maintains the union between the antigen and antibody molecules. Avidity is the overall and noncovalent attractive forces. strength of attachment of several antigen-antibody reactions and depends on the affinity  CHAPTER 1 n Immunology: Basic Principles and Applications in the Blood Bank 9 A Primary Secondary antibody response antibody response First Repeat exposure exposure IgG Plasma IgG cells IgM Plasma cells Amount of antibody in peripheral lymphoid tissues Activated Low-level B cells antibody production Plasma cells in bone Memory Plasma cells marrow B cell Memory in bone marrow B cell Naive B cell 0 5 10 >30 0 5 10 >30 Days after antigen exposure Days after antigen exposure B Primary response Secondary response Lag after Usually 5-10 days Usually 1-3 days immunization Peak Smaller Larger response Antibody Usually IgM>IgG Relative increase in IgG and, under isotype certain situations, in IgA or IgE (heavy chain isotype switching) Antibody Lower average affinity, Higher average affinity affinity more variable (affinity maturation) Fig. 1-6 Primary and secondary immune responses. The initial exposure to an antigen elicits the formation of IgM, followed by IgG antibodies and memory B cells. The second response to the same antigen causes much greater production of IgG antibodies and less IgM antibody secretion. (From Abbas AK, Lichtman AH: Basic immunology, ed 3, Philadelphia, 2011, Saunders.) Antigen-antibody reactions Applying the are reversible Law of Mass Action Ab Ag AbAg Equilibrium constant or affinity, K, is given by [AbAg] K K [Ab] [Ag] Fig. 1-7 Kinetics of antigen-antibody reactions. The ratio of the forward and reverse reaction rates gives the equilibrium constant. Ab, antibody; Ag, antigen. 10 PART I n Foundations: Basic Sciences and Reagents Good fit: high attraction, low repulsion Poor fit: low attraction, high repulsion Fig. 1-8 Good fit. A good fit between the antigenic determinant and the binding site of the antibody molecule results in high attraction. In a poor fit, the forces of attraction are low. TABLE 1-3 Forces Binding Antigen to Antibody Electrostatic forces Attraction between two molecules on the basis of opposite charge; (ionic bonding) a positively charged region of a molecule is attracted to the negatively charged region of another molecule Hydrogen bonding Attraction of two negatively charged groups (X−) for a H+ atom Hydrophobic bonding Weak bonds formed as a result of the exclusion of water from the antigen-antibody complex van der Waals forces Attraction between the electron cloud (−) of one atom and the protons (+) within the nucleus of another atom of the antibody, valency of the antigen, and noncovalent attractive forces. The goal of laboratory testing procedures in the blood bank is to create optimal conditions for antigen-antibody binding to facilitate the detection and identification of antibodies and antigens. SECTION 2 CHARACTERISTICS ASSOCIATED WITH RED CELL ANTIGEN-ANTIBODY REACTIONS Before discussing red cell antigens and antibodies, the reader must have a solid knowledge of the location of these concepts. When a blood sample undergoes centrifugation, the denser red cells travel to the bottom of the tube. The liquid portion of the sample is known as plasma (if an anticoagulant was added) or serum (no anticoagulant added and sample is allowed to clot). Red cell antigens are located on the red cells. They are part of the cell membrane or protrude from the cell membrane. Red cell antibodies are molecules in the plasma or serum (Fig. 1-9). RED CELL ANTIGENS Researchers have defined 30 blood group systems with more than 250 unique red cell antigens.3 A blood group system is composed of antigens that have been grouped accord- ing to the inheritance patterns of many blood group genes. Every individual possesses a unique set of red cell antigens. Because of a diversity of blood group gene inheritance patterns, certain racial populations may possess a greater prevalence of specific red cell antigens. Scientific research has determined the biochemical characteristics of many red cell antigens and their relationship to the red cell membrane. In biochemical terms, these antigens may take the form of proteins, proteins coupled with carbohydrate molecules  CHAPTER 1 n Immunology: Basic Principles and Applications in the Blood Bank 11 BLOOD SAMPLE Name_Date ID #_Initial Buffy coat has white blood cells and platelets. Antibodies are in plasma or serum. An